EC are constantly exposed to high levels of lipoprotein lipids, yet do not become lipid-laden. We have recently discovered that EC have a dramatic ability to efflux cholesterol, most likely to preserve endothelial integrity. Aortic EC express ABCG1 and ABCA1, two key cholesterol transporters. ABCG1 is a primary regulator of cholesterol efflux to HDL in reverse cholesterol transport. We have found a 70% reduction in ABCG1 expression in diabetic mouse aortic EC, causing a significant reduction in cholesterol efflux, and leading to increased EC cholesteryl ester content. In the current application, we hypothesize that chronic hyperglycemia in diabetes decreases EC ABCG1 function, thereby disturbing cholesterol homeostasis in the EC, and triggering endothelial activation. Endothelial activation and monocyte:endothelial interactions in the vessel wall are key initiating events in atherosclerotic plaque formation. We hypothesize that initiation of atherosclerosis is increased in the setting of Type 2 diabetes due to endothelial activation caused by a reduction in endothelial ABCG1 activity. Specific Aim 1 will test the hypothesis that ABCG1 expression regulates activation of EC by maintaining endothelial cholesterol homeostasis. We will utilize approaches in vitro to directly test the role of ABCG1 on endothelial activation. Intracellular cholesteryl ester accumulation, cholesterol efflux, chemokine production, and monocyte:endothelial interactions will be measured. Specific Aim 2 will test the hypothesis that endothelial ABCG1 expression regulates atherosclerotic plaque initiation in diabetic mouse models in vivo. Low density lipoprotein receptor (LDLR)-deficient mice are atherosclerosis-susceptible mice that develop diabetes when fed a diabetogenic diet. We will perform atherosclerosis studies using a conditional knockout mouse in which ABCG1 is deleted from endothelial cells to test that endothelial ABCG1 expression is a critical determinant of atherosclerosis initiation. Specific Aim 3 will test the hypothesis that glucose regulates ABCG1 promoter activity through action of the transcriptional repressor Id3. Our preliminary data indicate that the transcription factor E47 activates the ABCG1 promoter, and the repressor Id3, inhibits this activation. We will also examine regulation of ABCG1 protein by fatty acids. If we find that ABCG1 is an important regulator of atherosclerosis in diabetes, therapies to upregulate ABCG1 expression in diabetic patients could prove beneficial for prevention of atherosclerosis. PUBLIC HEALTH RELEVANCE: Endothelial cells have a surprising capacity to effectively efflux cholesterol to maintain normal endothelial homeostasis. Cholesterol efflux is regulated by ABC transporters, including ABCA1 and ABCG1. We hypothesize that ABCG1 function is necessary for normal endothelial function; in the absence of ABCG1, endothelial cells become activated and pro-inflammatory, contributing to a pro-atherogenic phenotype. We will explore this hypothesis using endothelial-specific ABCG1-deficient mice in atherosclerosis studies in vivo.